The long term objective of this research are to define the mechanisms of centra replication processes common to many (+) strand RNA viruses, a large and morphologically diverse class of pathogens causing widespread and serious human and animal diseases. Recombinant DNA manipulation of infectious viral cDNA clones will be used together with unusually tractable in vivo and in vitro replication systems to study RNA replication, recombination, and encapsidation in advanced model systems based on the related bromoviruses BMV (brome mosaic virus) and CCMV (cowpea chlorotic mottle virus), which embody RNA replication genes and other features conserved across a remarkably wide range of important animal and plant viruses. The results obtained will enhance biochemical and genetic understanding of a broad range of viruses and should provide a basis for developing new antiviral strategies and more sophisticated use of RNA viruses in genetic engineering. Recent results demonstrate that combined studies of BMV, CCMV, and engineered BMV/CCMV hybrids provide rich opportunities to define the interactions of viral proteins with each other and with their viral RNA substrates. Accordingly, molecular genetic and biochemical strategies will be combined to delineate the functions of specific domains in the polymerase- and helicase-related viral proteins that direct RNA replication, and to define the nature and significance of newly revealed interactions between these proteins. Cis-acting RNA regulatory sequences and trans-acting viral protein regions involved in selecting proper templates for replication will be mapped and their interaction characterized. Designed modification of viral RNA replication proteins will be used to exploit these protein-protein and protein-RNA interaction characteristics to develop trans-acting repressors of viral replication. For viral RNA recombination, which is thought to be a byproduct of RNA replication, demonstrated recombination between bromovirus deletion mutants will be used to determine how RNA sequence/structure features influence local recombination frequency, and to test for recombination polarity. Further experiments will determine protein sequence/structure requirement for effective RNA binding by the highly basic, putatively helical N- terminal arm of bromovirus coat protein, test whether this arm confers encapsidation specificity, and search for encapsidation signals in bromovirus RNA.